Attention has been paid to arachidonic acid (5,8,11,14-eicosatetraenoic acid), dihomo-y-linolenic acid (8,11,14-eicosatrienoic acid) and eicosapentaenoic acid (5,8,11,14,17-eicosapentaenoic acid) because these acids can become precursors of eicosanoids such as prostaglandin, leukotriene and tromboxane, and because the acids themselves have physiological activities. For example, eicosapentaenoic acid has been marketed as a food and medicine on the basis of its antithrombotic preventive action or lipid-lowering action.
Furthermore, it has recently been disclosed that arachidonic acid is similar to docosahexaenoic acid in that it is contained in breast milk, and that arachidonic acid is useful for growing infants (“Advances in Polyunsaturated Fatty Acid Research,” Elsevier Science Publishers, 1993, pp261-264). It has further been disclosed that arachidonic acid is important in the growth of the height and of the brain of embryos (Proc. Natl. Acad. Sci. USA, 90, 1073-1077 (1993), Lancet, 344, 1319-1322 (1994)).
Methods for producing such acids as arachidonic acid, dihomo-γ-linolenic acid or eicosapentaenoic acid in higher yields than conventional methods have been developed using microorganisms belonging to the genus Mortierella (Japanese Examined Patent Publication (Kokoku) No. 7-34752, Japanese Unexamined Patent Publication (Kokai) No. 6-153970, Japanese Unexamined Patent Publication (Kokai) No. 8-214893, Chapter 4 in Industrial Applications of Single Cell Oil, ed. by D. J. Kyle and C. Ratledge, American Oil Chemists' Society, Illinois, 1992, WO96/21037, Japanese Examined Patent Publication (Kokoku) No. 7-22513, Japanese Examined Patent Publication (Kokoku) No. 7-12315, and Japanese Unexamined Patent Publication (Kokai) No. 1-243992).
However, the microorganisms belonging to the genus Mortierella which have been used in the methods each have a low resistance to a high glucose concentration. Fed-batch culture is therefore practiced currently in the actual industrial production by culturing with aeration and agitation, so that the glucose concentration is in the range of 2 to 4% by weight. Accordingly, the methods have the problem that the production steps become complicated. Moreover, the microorganism belonging to the genus Mortierella, subgenus Mortierella which has heretofore been used for producing arachidonic acid, dihomo-γ-linolenic acid or eicosapentaenoic acid evidently shows a low growth level of microorganism (dried weight of the microbial cells per medium), as compared with a microorganism belonging to the genus Mortierella, subgenus Micromucor which has been used for producing γ-linolenic acid. As a result, the production amount of lipid containing highly unsaturated fatty acids per medium has been low (for example, the growth level of Mortierella alpina belonging to the subgenus Mortierella is 22.5 g/L, whereas that of Mortierella ramanniana var. angulispora belonging to the subgenus Micromucor reaches 79 g/L (Chapter 5, Chapter 7 in Industrial Applications of Single Cell Oil, ed. by D. J. Kyle and C. Ratledge, American Oil Chemists' Society, Illinois, 1992)).
The productivity of arachidonic acid by a microorganism belonging to the genus Mortierella, subgenus Mortierella has heretofore been disclosed, for example, as explained below. Japanese Unexamined Patent Publication (Kokai) No. 6-15397 discloses in an example that the microorganism produced 4.09 g/L of arachidonic acid after culturing for 7 days while the initial glucose concentration had been determined to be 2%. Moreover, Japanese Unexamined Patent Publication (Kokai) No. 8-214893 discloses in an example that the microorganism produced 2.3 g/L of arachidonic acid after culturing for 3 days while the initial glucose concentration had been determined to be 4.3%.
An example in which the concentration of a carbon source was increased will be explained below. The experimental results in chapter 4 of “Industrial Applications of Single Cell Oil, ed. by D. J. Kyle and C. Ratledge, American Oil Chemists' Society, Illinois, 1992” are as follows: 1.5 g/L of arachidonic acid was produced after culturing for 7 days while the initial dextrose concentration had been determined to be 9.8%; and 9.1 g/L of arachidonic acid was produced after culturing for 16 days while the initial dextrose concentration had been determined to be 9.8%. The growth of the microorganism was particularly poor due to a high initial dextrose concentration, and the production amount of arachidonic acid after culturing for 7 days was as low as only 1.5 g/L; the culturing period was determined to be 16 days for the purpose of ensuring the production amount.
Furthermore, in an example of WO96/21037, 5.3 g/L of arachidonic acid was produced after culturing for 8 days while the initial glucose concentration had been determined to be 10%. However, in order to suppress the inhibition of the growth of the microorganism caused by the high glucose concentration, procedures such as pH control and addition of salts were fully utilized, and complicated operations were required.
As explained above, for the production of substances by fermentation using microorganisms, in general, procedures such as a procedure in which the growth amount of the microorganism is increased and a procedure in which the production amount per microorganism is increased are taken when the productivity is intended to be enhanced. Increasing the concentration of the carbon source which is to become the nutrition source for growing the microorganism leads the microorganism to increase the growth amount, and culturing at a carbon source concentration as high as possible is preferred. On the other hand, however, increasing the concentration of the carbon source produces a harsh condition for growing the microorganism due to the resultant osmotic pressure, and results in suppressing the growth thereof.
As described above, the example wherein growth of the microorganism becomes poor when the initial glucose concentration is increased, and the production amount of arachidonic acid is as low as about 1.5 g/L after culturing for 7 days, and the example wherein the medium is adjusted by procedures such as controlling the pH and adding salts for the purpose of increasing the initial glucose concentration can also be regarded as examples showing difficulties of producing a substance by fermentation using a microorganism.
Accordingly, it has been desired to develop a method for producing arachidonic acid, dihomo-γ-linolenic acid or eicosapentaenoic acid efficiently with simple operations and in a large amount by finding a microorganism which is resistant to a carbon source of high concentration at the starting stage and which shows a sufficient growth level even in a medium having a high initial glucose concentration, and using the microorganism.